The ability of experimentalists to perturb biological systems has traditionally been limited to rigid pre- programmed (open loop) protocols. In contrast, real-time control allows the researcher to dynamically probe a biological system with parameter perturbations that are calculated functions of instantaneous system measurements (closed loop), thereby providing the ability to address diverse unanswered questions that are not amenable to traditional approaches. While real-time control applications are abundant throughout biological research, including, e.g., dynamic probing of ion-channel function in neurons and cardiac cells, adoption of such approaches lags. Unfortunately, for a number of technical reasons, real-time control is not possible with standard computer operating systems and software. Furthermore, commercial real-time systems are costly and often tailored for industrial applications. To circumvent these limitations, we developed a fast and highly versatile real- time biological experimentation system known as the Real-Time eXperiment Interface (RTXI). Based on Real-Time Linux, RTXI is open source and free, can be used with an extensive range of experimentation hardware, and can be run on Linux or Windows computers (when temporarily booted into Linux using an RTXI LiveCD). Importantly, RTXI has been adopted by many prominent scientific groups and has become an invaluable part of their scientific programs. In addition to the need to update and maintain RTXI for those, and future, end users, there remain important development avenues that would significantly expand its functionality and broaden its utility for the biological research community. Thus, for this competitive renewal, we propose: 1. To keep RTXI on the cutting edge by periodically updating its base code and core modules. 2. To enable new experiment paradigms. 3. To use RTXI's module architecture as the foundation for new protocol classes and application suites. 4. To improve the level of user support and documentation. The work proposed here would help ensure that RTXI not only remains a valuable research tool for a varied group of biological scientists, but that its utility, and the experiments it enables, continue to expand.